Functional connectivity between tRNA binding domains in glutaminyl-tRNA synthetase

JOURNAL OF MOLECULAR BIOLOGY

卷:256

Article

Sherman, JM ; Thomann, HU ; Soll, D

关键词: aminoacyl-tRNA synthetase;    tRNA specificity;    mutation;    anticodon;   

DOI  :  10.1006/jmbi.1996.0128

来源: Elsevier

【 摘 要 】

The structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNA(Gln) and ATP has identified a number a sequence-specific protein-tRNA interactions. The contribution to glutamine identity has previously been determined for the nucleotides in tRNA(Gln). Here, we report the mutational analysis of residues in all three tRNA recognition domains of GlnRS, thus completing a survey of the major sequence-specific contacts between GlnRS and tRNA(Gln). Specifically, we analyzed the GlnRS determinants involved in recognition of the anticodon which is essential for glutamine identity and in the communication of anticodon recognition to the acceptor binding domain in GlnRS. A combined in vivo and in vitro approach has demonstrated that Arg341, which makes a single sequence-specific hydrogen bond with U35 in the anticodon of tRNA(Gln), is involved in initial RNA recognition and is an important positive determinant for this base in both cognate and non-cognate tRNA contexts. However, Arg341, as well as Arg402, which interacts with G36 in the anticodon, are negative determinants for non-cognate nucleotides at their respective positions. Analysis of acceptor-anticodon binding double mutants and of a mutation of Glu323 in the loop-strand-helix connectivity subdomain in GlnRS has further implicated this domain in the functional communication of anticodon recognition. The better than expected activity (anticooperativity) of these double mutants has led us to propose an ''anticodon-independent'' mechanism, in which the removal of certain synthetase interactions with the anticodon eliminates structural constraints, thus allowing the relaxed specificity mutants in the acceptor binding domain to make more productive interactions. (C) 1996 Academic Press Limited

【 授权许可】

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